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Method of enhancing lysosomal alpha-Galactosidase A

a technology of lysosomal alpha-galactosidase and lysosomal alpha-galactosidase, which is applied in the direction of biocide, drug composition, cardiovascular disorder, etc., can solve the problem of excessive degradation

Inactive Publication Date: 2001-08-14
MT SINAI SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

It is a further object of the invention to provide a method of enhancing .alpha.-Gal A activity in mammalian cells, particularly in human cells. The methods of the present invention enhance the activity of both normal and mutant .alpha.-Gal A, particularly of mutant .alpha.-Gal A which is present in certain forms of Fabry disease.

Problems solved by technology

Furthermore, the majority of the mutant enzyme protein in cells formed aggregate in endoplasmic reticulum (ER) and was quickly degraded (7), suggesting that the deficiency of the enzyme activity in this mutant maybe primarily caused by the unsuccessful exit of ER leading to excessive degradation of the enzyme protein.

Method used

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  • Method of enhancing lysosomal alpha-Galactosidase A
  • Method of enhancing lysosomal alpha-Galactosidase A
  • Method of enhancing lysosomal alpha-Galactosidase A

Examples

Experimental program
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example 2

DGJ, the strongest inhibitor in vitro and most effective intracellular enhancer, was chosen for more detailed characterization. DGJ was added to the TgM or TgN fibroblasts (FIG. 2A) and lymphoblasts derived from Fabry patients with genotypes of R301Q or Q279E mutations (FIG. 2B). The enzyme activity found in TgM fibroblasts increased 6-fold by co-cultivation with 20 .mu.M DGJ and reached 52% of normal. The DGJ also showed a similar effect on lymphoblasts in which the residual enzyme activity was enhanced by 8- and 7-fold in R301Q and Q279E, i. e., 48% and 45% of normal. The enzyme activity in Tg normal (TgN) fibroblasts and normal lymphoblasts also showed an increase by cultivation with DGJ.

example 3

The TgM fibroblasts and human lymphoblasts of normal and patient with a mutation on R301Q were cultured in the presence of DGJ at 20 .mu.M. In the cultures without DGJ, the .mu.-Gal A activity in TgM fibroblasts or mutant lymphoblasts was unchanged (FIG. 3). However, by including DGJ, the enzyme activity showed significantly increase in these cell cultures. The enzyme activity in mutant lymphoblasts reached to 64% of those found in normal lymphoblasts cultured without DGJ at the fifth day. The enzyme activity in normal lymphoblasts was also enhanced 30% after cultivation with DGJ.

example 4

DGJ concentration dependence of .alpha.-Gal A enhancement in transfected COS-1 cells, TgM fibroblasts and lymphoblasts with a phenotype of R301Q was examined.

As shown in FIG. 4, the enzyme activity increased with the increase in the concentration of DGJ in the range of 0.2-20 .mu.M in transfected COS-1 cells (FIG. 4A) and lymphoblasts (FIG. 4C), and between 0.2-200 .mu.M in TgM fibroblasts (FIG. 4B), respectively. A higher concentration of DGJ suppressed the enhancement effect.

DE-HNJ showed the same effect on the enhancement of .alpha.-Gal A in COS-1 cells transfected with a mutant cDNA of the enzyme (R301Q) at the higher concentrations (1-1000 .mu.M) compared with DGJ (FIG. 5). It is clear that DE-HNJ at 1 mM in culture medium did not inhibit intracellular enzyme activity of COS-1 cells.

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Abstract

A method of enhancing the activity of lysosomal alpha-Galactosidase A (alpha-Gal A) in mammalian cells and for treatment of Fabry disease by administration of 1-deoxy-galactonojirimycin and related compounds.

Description

1. Field of the InventionThis invention relates to a method of enhancing the activity of lysosomal .alpha.-Galactosidase A (.alpha.-Gal A) in mammalian cells and for treatment of glycosphingolipid storage diseases, in particular Fabry disease, by administration of 1-deoxy-galactonojirimycin and related compounds.2. Background InformationFabry disease (1) is a glycosphingolipid lysosomal storage disease caused by an X-linked inherited deficiency of lysosomal .alpha.-galactosidase A (.alpha.-Gal A), an enzyme responsible for the hydrolysis of terminal .alpha.-galactosyl residue from glycosphingolipids. A deficiency in the enzyme activity results in a progressive deposition of neutral glycosphingolipids, predominantly globotriaosylceramide (ceramide trihexoside, CTH), in vascular endothelial cells causing renal failure along with premature myocardial infarction and strokes in patients with this condition (2). This disorder is classified by clinical manifestations into two groups: a cla...

Claims

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Application Information

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IPC IPC(8): A61K31/7008C12N9/40A61K31/445A61K31/4465A61K31/7042A61K31/7052A61K31/706A61P43/00C07D207/12C07D211/46C07H17/02
CPCA61K31/445C07D451/06C12N9/2465A61K31/7008A61K31/70A61P13/12A61P43/00A61P9/10
Inventor FAN, JIAN-QIANGISHII, SATOSHI
Owner MT SINAI SCHOOL OF MEDICINE
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